American Journal of Essential Oils and Natural Products 2017; 5(3): 01-06

ISSN: 2321-9114 AJEONP 2017; 5(3): 01-06 Chemical composition and in vitro antibacterial activity © 2017 AkiNik Publications Received: 02-05-2017 of essential oil from africanum Oliv. & Accepted: 04-06-2017 Hiern Philippe Babady-Bila Bila Nutraceuticals, 650 Portland Street Market, Philippe Babady-Bila, Dorothée Tshilanda Dinangayi, Damien Sha- Dartmouth, NS B2W 6P3, Tshibey Tshibangu, Emmanuel Lengbiye, Jean-Paul Ngbolua and Pius Canada Mpiana Tshimankinda Dorothée Tshilanda Dinangayi Department of Chemistry, Abstract University of Kinshasa, Kin. XI A hydrodistilled oil from the aerial part of Eupatorium africanum Oliv. & Hiern collected from D.R. Kinshasa, D.R. Congo Congo was analyzed by GC and GC/MS. Thirty three compounds representing 97.34% of the oil were

Damien Sha-Tshibey Tshibangu identified. The major components were -eudesmol (49.1%), 10-epi--eudesmol (11.30%), -humulene Department of Chemistry, (8.9%), hedycaryol (8.9%), and elemol (5.2%). The antibacterial activity of the essential oil and its University of Kinshasa, Kin. XI dominant constituent was evaluated against four bacteria strains (Staphyloccocus aureus ATCC 25952, Kinshasa, D.R. Congo Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Proteus vulgaris ATCC 4635) using the micro-dilution method. The results indicate that the two samples are active against all tested Emmanuel Lengbiye microorganisms. The oil has a moderate activity against Staphylococcus aureus and Pseudomonas Department of Biology, aeruginosa with a minimum inhibitory concentration (MIC) value of 125 µg/mL. The isolated major University of Kinshasa, Kin. XI constituent (-eudesmol) exhibits the highest activity against Staphylococcus aureus and Pseudomonas Kinshasa, D.R. Congo aeruginosa with a MIC value of 62.5 µg/mL. Both samples exhibited a MIC value of 250 μg/ml against Escherichia coli. and Proteus vulgaris. Jean-Paul Ngbolua Department of Biology, Keywords: Eupatorium africanum Oliv. & Hiern, , essential oil, chemical composition, University of Kinshasa, Kin. XI Kinshasa, D.R. Congo biological activity.

Pius Mpiana Tshimankinda 1. Introduction Department of Chemistry, Eupatorium africanum Oliv. & Hiern. [syn. Stomatanthes africanus (Oliv. & Hiern) R.M. University of Kinshasa, Kin. XI King & H. Rob] belongs to the genus Stomatanthes, Asteraceae family, tribe. This Kinshasa, D.R. Congo genus comprises 17 species distributed in (13 species) and in Africa (4 species). But according to Grossi, the genus Stomatanthes would be formed by only three of the four African species (S. africanus, S. meyeri and S. helenae). Stomatanthes zambiensis, the fourth African species, is excluded from Stomatanthes genus. It would be related with Eupatorium sensu stricto [1].

This is a perennial herb (sub-shrub 20–120 cm high) of hilly grassland throughout the [2] region from Guinea to West Cameroon and widespread in tropical Africa . It is used as herbal medicine against headache, diarrhea, dysentery and naso-pharyngeal affections [2, 3]. In Congo, a leaf decoction is made as a mouthwash for aphthera, and a tisane of the boiled root is used as a cough medicine and an anti-diarrhetic [4].

Phytochemical investigations and chemical composition of Eupatorium species essential oils (including many species now reclassified into other closely related genera) have been carried out by several researchers. These studies revealed the presence of a variety of monoterpernes, sesquiterpenes, sesquiterpene lactones, flavonoids, phenolic and acetylenic compounds, triterpenes, and alkaloids with cytotoxic, antitumoral, antimicrobial, antioxidant and anti- [5-6] inflammatory activities has been reported . A review of antimicrobial activity of the genus Eupatorium L. (Asteraceae) has been recently published and Eupatorium africanum is not mentioned in this paper [7]. The following terpenoids were reported as major constituents of Correspondence: Eupatorium species essential oils: α-pinene (50.98 %), -pinene (10.5%) sabinene (46.7%), Philippe Babady-Bila limonene (9.63-23.3%), camphene (8.9%), (+)-camphor (15.46%), 2,5-dimethoxy-p-cymene Bila Nutraceuticals, 650 Portland Street Market, or thymohydroquinone dimethyl ether (20.8-60.7%), thymol, p-cymene (23.7%), thymol Dartmouth, NS B2W 6P3, methyl ether (36.3%), aristolone+laevigatin (23.6%), globulol (16.2-25.1%), caryophyllene Canada oxide (17.4–30.1%), germacrene D (8.6–21.6%), spathulenol (14.2%), (-)-spathulenol

~ 1 ~ American Journal of Essential Oils and Natural Products

(0.48-25.16 %), α-zingiberene (57.5%), α-gurjunene (19.5%), operated at an ionization energy of 70 eV with a mass scan (E)-β-bisabolene (9.7%), α-selinene (9.0%), β-caryophyllene range of 40-400 amu. (1.11-41.7%), ocimene (4.78 %), δ-elemene (10.57%), α- humulene (0.25-14.6%), patchoulene (9.24%), viridiflorol 2.5 Identification of oil constituents (9.16%), γ-elemene (5.92%), (+)-δ-cadinene (5.83%), (-)-δ- The identification of constituents was based on a comparison cadinol (2.67%), α-santalene (2.53%), β-cubebene (1.64%), of their retention indices and mass spectra with spectral data (+)-nerolidol (1.63%), selina-4(15),7(11)-dien-8-one (36.6%), from several sources, by searching in the GC-MS library, and -muurolene (10.4-19%), myrcene (15.7%), valencene by matching their fragmentation patterns in mass spectra with (10.5%), cyperone (16.9%), and pregeijerene (14.3%), those of published [24-28]. The percentage of the individual phellandrene (3.85%), α-bisabolol (9.53%), bornyl acetate constituent was obtained from FID area percentages without (8.98-15.6%), -bisabolene (6.16%), amorph-4-en-7-ol the use of correction factors. Retention indices were (9.6%), 3-acetoxyamorpha-4,7(11)- dien-8-one (7.8%) and calculated relative to C8-C24 n-alkanes, and compared with amorph-4,7(11)-dien-8-one (5.7%), neryl isobutyrate (17.6%). values reported in the literature [24-26]. The major non-terpenoid compounds reported were methyl chavicol (42.2%) and 1-naphthalenol (17.50%). 2.6 Antibacterial Activity Amorph-4-en-7-ol (9.6%), 3-acetoxyamorpha - 4,7(11) - dien- 2.6.1 Microorganisms 8-one (7.8%) and amorph-4,7(11)-dien-8-one (5.7%) were Four strains of microorganisms from the American Type identified in Eupatorium adenophorum oil. Amorphene Culture Collection (ATCC, Rockville MD, USA) were used derivatives (19.8–41.4%) may be considered as characteristic in the present study: Staphylococcus aureus (S. aureus ATCC constituents of E. adenophorum [5, 8-23]. 25952), Escherichia coli (E. coli ATCC 27195), The present investigation reports the results of GC and GC- Pseudomonas aeruginosa (P. aeruginosa ATCC 9027), and MS analyses of the essential oil from the leaves of Proteus vulgaris (P. vulgaris ATCC 4635) strains. Eupatorium africanum Oliv. & Hiern growing wild in D.R. Congo, and its antibacterial activity evaluation. To the best of 2.6.2 Determination of Minimum inhibitory concentration our knowledge, this is the first report on the chemical (MIC) composition and the biological activity of the The antibacterial activity of Eupatorium africanum essential Eupatorium africanum essential oil. oil and its major constituent was assessed against selected bacteria strains by the micro-well dilution method and the 2. Materials and methods minimum inhibitory concentration (MIC) values, which 2.1 Plant material represent the lowest sample concentrations that completely The Eupatorium africanum leaves were collected in the inhibit the growth of microorganisms were obtained using this Bombo-Lumene Game reserve, Kinshasa Province, D.R. method [29]. Congo, and authenticated by a voucher specimen (H. The 10 mg samples (essential oil and isolated major BREYNE 2785) kept at the INERA herbarium, Department of constituent) were each dissolved in DMSO (250 μL) and Biology, University of Kinshasa, D.R. Congo. diluted with Mueller–Hinton Broth (MHB) in order to reach The collected fresh leaves were dried at room temperature in a concentrations of 2000 μg/mL and a 5 ml solution (final well-ventilated room and preserved in tightly closed bumper volume, and 5% DMSO final concentration). These solution bags at room temperature in the absence of light. were used as stock solutions. The inocula of microorganisms were prepared from 24h old 2.2 Isolation of essential oil MHB cultures. The microbial suspensions were prepared by Dried plant material (300 g) was subjected to hydro- adding five colonies of each of the test bacteria to 2 mL of distillation for 4 hours using a Clevenger type apparatus to with sterile physiological solution (0.9% NaCl) and adjusted produce an essential oil at a yield of 0.4%. The oil was dried with this sterile physiological solution to match that of a 0.5 over anhydrous sodium sulfate and stored in sealed vials at McFarland standard solution (108 cells/ml). They were then low temperature (4 C). diluted (1/100) to achieve 106 CFU/mL. The assay was carried out using sterile clear polystyrene 96- 2.3 Gas Chromatographic (GC) Analysis well microtiter plates (round bottom). The wells in the GC analysis was performed using a Hewlett-Packard 5880A columns 2 to 8 and those in columns 11 and 12 were filled gas chromatograph equipped with a flame ionization detector with 100 µL MHB (Mueller Hinton Broth). Briefly 200 µL of (FID) and data-handling system. The GC was fitted with a stock solution of each Eupatorium sample were added to the DB-5 fused silica capillary column (30m × 0.25 mm, film wells in column 1 (A1 to H1), and two-fold serial dilutions thickness 0.25 μm). The oven temperature was programmed were made from column 1 to column 8. Then 5 µL of the as follows: 40 °C for 5 min, from 40 to 200 °C at a rate of 4 inoculum were dispensed to all the wells except those in °C/ min, and 10 oC/min. ramp to 300 oC. Helium was used as column 12. The wells in columns 11 and 12 we used as carrier gas at the flow rate of 1 mL/min. Injector and detector positive and negative controls. The negative control wells temperatures were maintained at 280 and 295 °C, (growth control) contained MHB and bacteria suspension respectively. The volume of sample injected was 0.2 L and without test sample (column 11) and the positive control wells the split ratio was 1:30. contained only MHB (control of MHB sterility: column 12). The microplates (96 wells) were incubated at 37°C for 2.4 GC/MS analysis 24 hours. After the incubation, 5 µL de colorant resazurin 1% GC/MS analysis was performed with Varian Saturn 2100T (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) were added to GC/MS fitted with a DB-5 fused silica capillary column (30m each well and the microplates were then incubated for 5 × 0.25 mm, film thickness 0.25 μm). The GC conditions were hours. The minimum inhibitory concentration (MIC) was as described for GC analysis. The mass spectrometer was determined as the lowest essential oil concentration at which no growth were observed after 24 and 48 hours. All ~ 2 ~ American Journal of Essential Oils and Natural Products

experiments were performed in triplicate. eight trace components (<0.03%) were not identified. The mass spectra (reported in Table 1) of the unidentified 3. Results & Discussion components(UI) UI 2, UI 3, UI 5 to UI 9 suggest that these The oil isolated by hydrodistillation of the Eupatorium compounds are polyoxygenated sesquitepenes. UI2 is a africanum leaves were found to be yellow and yields 0.40% sesquiterpenediol (expected MW=238). The peaks at m/z=223 + + + + (w/w), based on dry weights. Its composition is summarized (M -CH3), 220 (M -H2O), 207 (M -CH2OH), 205 (M -CH3,- + + in Table I. Constituents are listed in order of their elution H2O), 189 (M -H2O,-CH2OH), 177 (M -CH2OH, -2CH3) from a DB-5 capillary column and were identified by GC/MS account for the presence of two -OH groups in the molecule, analysis in combination with retention indices. The main one of which is part of a -CH2OH group. Unidentified constituent was isolated and identified as -eudesmol. constituents UI3, UI5 to UI9 are monoacetylated This study reports the presence of 42 components, 33 of sesquiterpenetriols (expected MW=296). They all show a + which were identified and quantified, representing 97.34% of peak at m/z=281 corresponding to M -CH3. The peak at the total Eupatorium africanum oil composition. This oil was m/z=59 in UI3 (base peak), UI6 (90%) and UI7 (base peak) constituted mainly by oxygenated sesquiterpenes (82.64%). suggests the presence of a hydroxyisopropyl group in these Sesquiterpene hydrocarbons represented 12.77%) and compounds. UI8 and UI9 mass spectra exhibit the similar monoterpenes accounted only for 1.93% of the oil. - fragmentation patterns with different relative intensity for Eudesmol made up the largest component of the oil (49.1%) corresponding peaks. These two components might be and the second largest component was 10-epi--eudesmol isomers. Although we did not identify most of the unknown (11.30%). Other major constituents (> 5.0 %) of the oil were constituents of the Eupatorium africanum oil, it is worth hedycaryol (8.9%), -humulene (8.9%) and elemol (5.2%). nothing that this is the first report indicating the presence of The minor constituents (< 5.0 and > 0.03 %) of the oil were polyoxygenated sesquiterpenes in Eupatorium species eremoligenol (1.90%), allo-spathulenol (1.90%), humulene essential oil. oxide II (1.70%), (E)--caryophyllene (1.47%), limonene Previously reported biologically active sesquiterpene lactones, (1.10%), -cadinene (0.86%), 4-epi-cubebol (0.63%), cadinenes, chromenes and thymol derivatives were not detected in present investigation. The result of this caryohyllene oxide (0.63%), myrcene (0.47%), -cubenene investigation suggests that the essential oil of (0.47), cubebol (0.44%), germacrene D (0.37%), valencene Eupatorium africanum can serve as a source of -eudesmol (0.33%), (E)--ocimene (0.32%), -eudesmol (0.31%) and that constitutes 49.1% of the oil. trans-cadina-1(6),4-diene (0.27%). One minor (0.31%) and

Table 1: The chemical composition of Eupatorium africanum essential oil

No Componentsa RIb (%) Identification 1 Tricyclene 925 0.04 A, B 2 -Fenchene 951 trc A, B 3 Sabinene 972 tr A, B 4 -Pinene 981 tr A, B 5 Myrcene 993 0.47 A, B 6 Limonene 1032 1.10 A, B 7 (E)--Ocimene 1041 0.32 A, B 8 Linalool 1091 tr A, B 9 -Elemene 1330 0.06 A, B 10 -Ylangene 1365 0.04 A, B -Cubebene 1382 0.47 A, B 12 UI1 1386 0.03 13 7-epi-sesquithujene 1392 tr A, B 14 (E)--Caryophyllene 1412 1.47 A, B 15 trans-Muurola-3,5-diene 1453 tr A, B 16 -Humulene 1459 8.90d A, B 17 trans-Cadina-1(6),4 diene 1471 0.27 A, B 18 Germacrene-D 1474 0.37 A, B 19 4-epi-Cubebol 1487 0.63 A, B 20 γ-Amorphene 1492 0.33 A, B 21 Cubebol 1510 0.44 A, B 22 -Cadinene 1524 0.86 A, B 23 Zonarene 1526 tr A, B 24 Hedycaryol 1542 8.90 A, B 25 Elemol 1551 5.83 A, B 26 Caryophyllene oxide 1581 0.63 A, B 27 Humulene oxide II 1611 1.70 A, B 28 Allo-spathulenol 1621 1.90 A, B 29 10-epi--Eudesmol 1624 11.30 A, B 30 Eremoligenol 1631 1.90 A, B 31 -Eudesmol 1650 49.1 A, B 32 -Eudesmol 1654 0.31 A, B 33 UI2 (Sesquiterpenediol) 1738 tr 34 -Costol 1773 tr A, B ~ 3 ~ American Journal of Essential Oils and Natural Products

35 Eudesma-11-en-4,6-diol 1799 tr A, B 36 UI3 (Monoacetylated sesquiterpenetriol) 1882 tr 37 UI4 (Sesquiterpene alcohol: (acetylated?)) 2031 tr 38 UI5 (Monoacetylated sesquiterpenetriol) 2046 tr 39 UI6 (Monoacetylated sesquiterpenetriol) 2105 tr 40 UI7 (Monoacetylated sesquiterpenetriol) 2183 tr 41 UI8 (Monoacetylated sesquiterpenetriol) 2395 0.31 42 UI9 (Monoacetylated sesquiterpenetriol) 2472 tr

Total identified 97.34% AcO-), 191(8), 177(2), 161(1), 141(2), 131(5), 107(2), 93 Group Components (5), 90(11), 80 (28), 78 (30), 76 (20), 73(8), 67 (22), 55 • Monoterpene hydrocarbons : 1.93% (34), 43 (100). • Sesquiterpene hydrocarbons : 12.77%  UI9 (Monoacetylated sesquiterpenetriol): 281 (44, M+- + + • Oxygenated sesquiterpenes : 82.64% CH3), 267 (15), 265 (11, M -CH2OH), 207 (96, M - - a. Compounds listed in order of elution from DB-5 column 2CH3,-AcO ), 190 (21), 161 (25), 149 (4), 131(15), 121 b. RI: retention index relative to n-alkanes (C8–C20) on the (13), 119 (27), 105 (27), 93 (14), 90 (28), 86 (31), 84 DB-5 column. (37), 80 (27), 78 (14), 76(15), 72 (22), 67 (14), 55 (16), c. Traces: (<0.03%) 50 (29), 49 (66), 47 (45), 43 (100) d. Italic/Boldface designates the major constituents A: RI The results of the evaluation of antibacterial activity of the B: MS essential oil and its main constituent are reported in Table 2. The oil exhibited moderate antibacterial activity against Mass spectra of unidentified constituents: m/z (relative Staphylococcus aureus and Pseudomonas aeruginosa with intensity %) MIC values of 125 µg/mL and a weak antibacterial activity  UI1: 174 (100, M+), 161 (1), 142 (1), 137(1), 121 (3), 105 against Escherichia coli and Proteus vulgaris with MIC (1), 93 (5), 91(3), 83 (3), 81(1), 55 (3), 51 (3), 49 (7), values of 250 µg/mL while the main constituent of the oil 41(3) showed a strong antibacterial activity against Staphylococcus + +  UI2 (Sesquiterpenediol: 223 (30, M -CH3), 220 (13, M - aureus and Pseudomonas aeruginosa with MIC values of + + H2O), 207 (21, M -CH2OH), 205 (15, M -CH3,-H2O), 62.5 µg/mL, and a weak antibacterial activity against + + 189 (8,M -H2O,-CH2OH), 177 (10, M -CH2OH, -2CH3), Escherichia coli and Proteus vulgaris with MIC values of 161 (18), 159 (16), 149 (8), 141 (22), 137 (15), 124 (20), 250 µg/mL. 123 (25), 121 (18), 107 (25), 95 (19), 93 (18), 90 (20), 80 Several studies report that -eudesmol exhibit multiple (22), 79 (26), 77 (18), 69 (39). 59 (17), 55 (54), 43 (100), pharmacological activities, including anti-angiogenic anti- 41(89) mutagenic, anti-tumor, antipeptic, antisalmonella, hepato-  UI3 (Monoacetylated sesquiterpenetriol): 281(7, M+- protective, sedative, antiepileptic, anticonvulsant activities, + + [30- CH3), 222 (8, M -CH3,-59), 205 (4, M -CH3,-59,-17), and its neuromuscular blocking effect of succinylcholine 191 (14), 189 (10), 180 (14), 175 (9), 162 (21), 147 (18), 35]. 135 (18), 125 (26), 121 (17), 109 (26), 107 (48), 105 (24), 96 (22), 95 (32), 93 (39), 91(25), 81 (40), 78 (28), Table 2: Antimicrobial activity of the Eupatoriun africanum 67 (44), 59 (100), 56 (33), 49 (39), 43 (74), 41(52). essential oil and its major constituent.

+  UI4 (Sesquiterpene alcohol: acetylated?): 207 (20, M - Samples concentrations (μg/mL) + CH3?), 164 (17, M -CH3,-43 ?), 161(17), 159 (7), 149 Microorganism (Gram) Essential Oil β-eudesmol (18), 147 (20), 124 (26), 119 (45), 109 (21), 107 (22), 250 125 250 62.5 105 (21), 95 (37), 93 (34), 91 (26), 81(49), 78 (42), 67 Escherichia coli (-) MIC MIC (22), 59 (49), 55 (41), 43 (96), 41(100). Staphylococcus aureus (+) MIC MIC  UI5 (Monoacetylated sesquiterpenetriol): 281 (39, M+- Pseudomonas aeruginosa (-) MIC MIC + Proteus vulgaris (-) MIC MIC CH3), 207 (43, M -CH3,-74), 162 (14), 148 (14), 142 (10), 130 (14), 128 (15), 121 (13), 108 (15), 107 (15), 103 (10), 93 (18), 90 (17), 83 (32), 80 (26), 79 (49), 4. Conclusions 67(18), 65 (22), 59 (55), 49 (66), 43(100). The chemical analyses of the Eupatorium africanum essential  UI6 (Monoacetylated sesquiterpenetriol): 281(37, M+- oil by GC/MS allowed the identification of 33 constituents, + + representing 97.34% of the total oil. The main constituent was CH3), 220 (24, M -17,-59), 207 (73, M -2CH3,-59), 202 (24, 220-H2O), 191 (27), 187 (58), 162 (25), 159 (36), -eudesmol (49.10%) and the yield of essential oils was 0.4%. 147 (44), 135 (22), 133 (20), 131 (25), 119 (38), 118 Oxygenated sesquiterpenes were the major components of the (23), 109 (19), 105(100), 95 (44), 90 (43), 80 (27), 78 oil (82.64%). Sesquiterpenes (12.77%) and monoterpenes (53), 76 (41), 72 (17), 69 (32), 67 (33), 62 (38), 59 (90), (1.93%) represented the minor constituents. Polyoxygenated 43(69). sesquiterpenes, although in very low concentrations, have  UI7 (Monoacetylated sesquiterpenetriol): 281 (37, M+- been detected in this oil. Aromatic compounds were not + present in this oil. The oil exhibited moderate antibacterial CH3), 221(14, M -CH3,-AcOH), 220 (10), 207 (47), 205 (8), 165 (10), 162 (24), 159 (7), 134 (21), 123 (10), 119 activity against Staphylococcus aureus and Pseudomonas (27), 109 (10), 107 (20), 105 (19), 95 (27), 93 (22), aeruginosa and a weak antibacterial activity against 91(14), 84 (20), 80 (25), 78 (18), 76 (4), 59 (100), 55 Escherichia coli and Proteus vulgaris. The major constituent (13), 53 (25), 49 (36), 43 (65). of the oil was isolated and identified. It showed a strong  UI8 (Monoacetylated sesquiterpenetriol): 281(17, M+- antibacterial activity against Staphylococcus aureus and + + Pseudomonas aeruginosa and a weak antibacterial activity CH3), 267(3), 265(3, M -CH2OH), 207 (32, M -2CH3,- ~ 4 ~ American Journal of Essential Oils and Natural Products

against Escherichia coli and Proteus vulgaris. Eupatorium adenophorum. Pharm Biol. 2010; 48(5):539- The present study represents the first comprehensive analyses 44. of the oil obtained from the leaves of Eupatorium africanum. 15. Umpiérrez ML, Santos E, Mendoza Y, Altesor P, Rossini C. Essential oil from Eupatorium buniifolium leaves as Acknowledgments potential varroacide Parasitol Res. 2013; 112(10):3389- The authors wish to thank Mr. Nlandu (Department of 400. Biology, University of Kinshasa D.R. Congo) for the 16. Pimienta-Ramírez L, García-Rodríguez YM, Ríos- authentication of the plant material and Dr. Dave Vadenais Ramírez EM, Lindig-Cisneros R, Espinosa-García FJ. (Nipissing University, North Bay, Ontario, Canada) for his Chemical composition and evaluation of the essential oil skillful technical assistance. from Eupatorium glabratum as biopesticide against Sitophilus zeamais and several stored maize fungi Journal References of Essential Oil Research. 2016; 28(2):113-120. 1. Grossi MA. Revisión sistemática, análisis cladístico y 17. Maia JGS, Zoghbi MGB, Andrade EHA, Silva M, Luz biogeografía del género Stomatanthes R.M. King & H. AIR, Silva JD. Essential oils composition Robinson (Asteraceae, Eupatorieae). PhD. Thesis, of Eupatorium species growing wild in the Amazon Facultad de Ciencias Naturales y Museo, Universidad Biochem. Sys. Ecol. 2002; 30:1071-1077. Nacional de La Plata, Argentina, 2010. 18. Lamaty G, Menut C, Zollo PHA, Kuiate JR, Bessiere JM, 2. Burkill HM. The useful of west tropical Africa, Ouamba JM et al. Aromatic Plants of Tropical Central Royal Botanic Gardens, Kew, Kew Publishing, 1985, 1. Africa. IV. Essential oils of Eupatorium odoratum from 3. http://www.biomedcentral.com/content/supplementary/17 Cameroon and Congo. J. Essen. Oil Res, 1992; 4:101-105. 46-4269-4-10-s2.pdf 19. Nguyen XD, Luu DC, Pham VK, Muselli A, Casanova J, 4. Bouquet A. Féticheurs et médecines traditionnelles du Barthel A et al. Volatile constituents of the stem and Leaf Congo (Brazzaville), Mémoire O.R.S.T.O.M, 1969, 94. oils of Eupatorium coelestinum from Vietnam. J. Essent. 5. Padalia RC, Ram S, Verma RS, Sundaresan V. Volatile Oil Res. 1998; 10:478-482. Constituents of Three Invasive Weeds of Himalayan 20. Pino JA, Rosado A, Fuentes V. Essential oil Region Rec. Nat. Prod. 2010; 4(2):109-114. of Eupatorium capillifolum small from Cuba. J. Essent. 6. Inya-agha SI, Oguntimein BO, Sofowora A, Benjamin Oil Res. 1998; 10:79-80. TV. Phytochemical and Antibacterial Studies on the 21. Bamba D, Bessiere JM, Marion C, Pelissier Y, Fouraste I. Essential Oil of Eupatorium odoratum International Essential oil of Eupatorium odoratum. Planta Med. 1993; Journal of Crude Drug Research. 1987; 25(1):49-52. 59:184-185. 7. Sobrinho ACN, Morais SM, Souza EB, Fontenelle ROS. 22. Elema ET, Schripsema J, Malingre TM. Flavones and The genus Eupatorium L. (Asteraceae): A review of their flavonol glycosides from Eupatorium cannabinum L. antimicrobial activity J. Med. Plants Res. 2017; 11(3):43- Pharm. Weekbl. Sci. 1989; 20:161-164. 57. 23. Albuquerque MRJR, Souza EB, Mesquita EF, Nunes EP, 8. Padalia RC, Bisht DS, Joshi SC, Mathela CS. Chemical Cunha AN, Silveira ER. Volatile constituents from leaves Composition of the Essential Oil from Eupatorium of Vernonia chalbaea Mart. and Eupatorium adenophorum Spreng Journal of Essential Oil Research. ballotaefolium H.B.K. J. Essent. Oil Res. 2001; 13:376- 2009; 21(6):522-524. 377. 9. Joshi RJ. Chemical Composition of the Essential oil 24. Adams RP. Identification of Essential Oil Components by of Chromolaena odorata (L.) R. M. King & H. Rob. Gas Chromatography/ Mass Spectrometry 4th Ed. Journal of Chemistry. 2013, 4. Article ID 195057 Allured Publishing Co. Carol Stream, IL, USA, 2007. 10. Paolini J, Costa J, Bernardini AF. Analysis of the 25. NIST. NIST Chemistry Web Book: NIST Standard essential oil from the roots of Eupatorium Reference Database Number 69. Available: cannabinum subsp. corsicum (L.) by GC, GC-MS http://webbook. nist.gov/chemistry/ Accessed 05 Jan and 13C-NMR Phytochemical Analysis, 2007; 18(3):235- 2016 244. 26. El-Sayed AM. The Pherobase: Database of Pheromones 11. Tabanca N, Bernier UR, Tsikolia M, Becnel J, Sampson and Semiochemicals, 2014. http://www.pherobase.com B, Werle C et al. Eupatorium capillifolium Essential Oil: Accessed 15 Jan 2016 Chemical Composition, Antifungal Activity, and 27. http://www.massbank.jp Accessed 15 Feb. 2016. Insecticidal Activity, Natural Product Communications, 28. Suzuki M, Kowata N, Kurosawa E. Epicubebol and 2010; 5(9):1409. related Sesquiterpenoids form Brown Alga Dictyopteris 12. Souza TJT. Determinanaçao de Composiçao Quimica E divaricate, Bull.Chem.Soc.Jpn. 1981; 54(8):2366-2368. Avaliaçao Preliminar das Atividades Antioxidante e 29. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro Anticolinesterásica dos Óleos Voláteisde Espécies de evaluating antimicrobial activity: A review. Journal of Eupatorium L. (Asteraceae), M.S. Thesis Faculdade de Pharmaceutical Analysis. 2016; 6:71-79. Farmáceuticas, Universidade de , 30. Wade D, Silveira A, Rollins-Smith L, Bergman T, 2007. Silberring J, Lankinen H. Hematological and antifungal 13. Lancelle HG, Giordano Oscar S, Sosa ME, Tonn CE. properties of temporin A and a cecropin A-temporin A Chemical composition of four essential oils from hybrid. Acta Biochim. Pol. 2001; 48:1185-1189. Eupatorium spp. Biological activities toward Tribolium 31. Tsuneki H, Ma EL, Kobayashi S, Sekizaki N, Maekawa castaneum (Coleoptera: Tenebrionidae) Rev. Soc. K, Sasaoka T et al. Antiangiogenic activity of β- Entomol. Argent. 2009; 68(3-4):329-338. eudesmol in vitro and in vivo. Eur. J Pharmacol. 2005; 14. Kurade NP, Jaitak V, Kaul VK, Sharma OP. Chemical 512(2-3):105-115. composition and antibacterial activity of essential oils of 32. Kimura M, Tanaka K, Takamura Y, Nojima H, Kimura Lantana camara, Ageratum houstonianum and I, Yano S et al. Structural components of β-eudesmol ~ 5 ~ American Journal of Essential Oils and Natural Products

essential for its potentiating effect on succinyl choline- induced neuromuscular blockade in mice. Biol. Pharm. Bull. 1994; 17(9):1232-1240. 33. Ma EL, Li YC, Tsuneki H, Xiao JF, Xia MY, Wang MW et al. β-eudesmol suppresses tumour growth through inhibition of tumour neovascularisation and tumour cell proliferation. J. Asian Nat. Prod. Res. 2008; 10(1-2):159- 167. 34. Britto AC, de Oliveira AC, Henriques RM, Cardoso GM, Bomfim DS, Carvalho AA et al. In vitro and in vivo antitumor effects of the essential oil from the leaves of Guatteria friesiana. Planta Med. 2012; 78(5):409-414. 35. http://www.rain-tree.com/manaca-activity Biological Activities for Extracts of Manacá (Brunfelsia grandiflora) Accessed 20 December 2016

~ 6 ~